Salmonella reduces tumor metastasis by downregulation C-X-C chemokine receptor type 4

Tumor metastasis is the main reason for the death of most cancer patients. C-X-C chemokine receptor type 4 (CXCR4) has been demonstrated to be overexpressed in numerous types of cancer. CXCR4 selectively binds with stromal cell-derived factor 1 (SDF1), also known as C-X-C family chemokine ligand 12 (CXCL12) (CXCL12/SDF-1), which induced tumor proliferation and metastasis. Recently, the use of conventional cancer treatments had some limitation; bacteria treatment for cancer becomes a trend that overcomes these limitations. Plenty of studies show that Salmonella has anti-tumor and anti-metastatic activity. The current study aimed to investigate Salmonella suppresses CXCR4 protein expression and tumor cell migration ability in B16F10 melanoma and LL2 lung carcinoma cells. Salmonella reduced CXCR4 protein expression through downregulating Protein Kinase-B (Akt)/Mammalian Target of Rapamycin (mTOR) signaling pathway. In cells transfected with constitutively active Akt plasmids, a reverse effect of Salmonella-induced inhibition of CXCR4 was observed. Tumor cells have chemotactic response to CXCL12 in migration assay, and we found that Salmonella reduced tumor chemotactic response after CXCL12 treatment. The C57BL/6 mice were intravenously injected with B16F10 and LL2 cells pre-incubated with or without Salmonella, the tumor size and lung weight of Salmonella group had obviously decreased, indicating anti-metastatic effect that confirmed the findings from the in vitro experiments.     

Salmonella alters heparanase expression and reduces tumor metastasis

Salmonella causes salmonellosis, is a facultative anaerobe and is one of the common Gram-negative bacteria. Salmonella has anti-tumor potential and tumor-targeting activity. The heparin sulfate on cell surfaces can be cleaved by heparanase that is an endo-β-D-glucuronidase. Heparanase can destroy the extracellular matrix and is involved in tumor metastasis and angiogenic activity. Previously, Salmonella was demonstrated to inhibit tumor metastasis. It remains unclear whether Salmonella inhibits metastasis by regulating heparanase. The expression of heparanase in Salmonella-treated tumor cells was found to be decreased. Transwell and wound-healing assays demonstrated the inhibition of cell migration after Salmonella treatment. Salmonella was found to influence the levels of phosphate-protein kinase B (P-AKT) and phosphate-extracellular regulated protein kinases (P-ERK), which are involved in heparanase expression. Salmonella reduced the heparanase expression induced upregulating PERK and PAKT signaling pathways. The mice bearing an experimental metastasis tumor model was used to evaluate the anti-tumor metastatic effects of Salmonella. Compared with the control group, Salmonella significantly reduced the number of metastatic nodules and enhanced survival. The results of our study indicate that Salmonella plays a vital role in the inhibition of tumor metastasis through the downregulation of heparanase.     

The Contribution of Cytolethal Distending Toxin to Bacterial Pathogenesis

Cytolethal distending toxin (CDT) is a bacterial toxin that initiates a eukaryotic cell cycle block at the G₂ stage prior to mitosis. CDT is produced by a number of bacterial pathogens including: Campylobacter species, Escherichia coli, Salmonella enterica serovar Typhi, Shigella dystenteriae, enterohepatic Helicobacter species, Actinobacillus actinomycetemcomitans (the cause of aggressive periodontitis), and Haemophilus ducreyi (the cause of chancroid). The functional toxin is composed of three proteins; CdtB potentiates a cascade leading to cell cycle block, and CdtA and CdtC function as dimeric subunits, which bind CdtB and delivers it to the mammalian cell interior. Once inside the cell, CdtB enters the nucleus and exhibits a DNase I-like activity that results in DNA double-strand breaks. The eukaryotic cell responds to the DNA double-strand breaks by initiating a regulatory cascade that results in cell cycle arrest, cellular distension, and cell death. Mutations in CdtABC that cause any of the three subunits to lose function prevent the bacterial cell from inducing cytotoxicity. The result of CDT activity can differ somewhat depending on the eukaryotic cell types affected. Epithelial cells, endothelial cells, and keratinocytes undergo G₂ cell cycle arrest, cellular distension, and death; fibroblasts undergo G₁ and G₂ arrest, cellular distension, and death; and immune cells undergo G₂ arrest followed by apoptosis. CDT contributes to pathogenesis by inhibiting both cellular and humoral immunity via apoptosis of immune response cells, and by generating necrosis of epithelial-type cells and fibroblasts involved in the repair of lesions produced by pathogens resulting in slow healing and production of disease symptoms. Thus, CDT may function as a virulence factor in pathogens that produce the toxin.     

The “Atypical” Mycobacteria: Recognition and Disease Association

Abstract

Current advances in the understanding of the pathogenicity of the agents of diarrheal infections, Vibrio cholerae, diarrheagenic E. coli, Shigella, Salmonella, and enteropathogenic Yersinia, have, to a great extent, become possible due to morphological studies of host-pathogen interactions in natural and experimental infections. Despite a multigenic nature and a diversity of pathogenic features in the bacterial species and even in serogroups of the same species, it is now possible to delineate four major patterns of interaction of enteric pathogens with their cellular targets, the enterocytes, and with the immune apparatus of the gut. These patterns, epicellular cytotonic, epicellular restructuring cytotonic, invasive intraepithelial cytotonic and cytotoxic, and invasive transcellular cytotonic and cytotoxic bacteremic, underlie early pathogenesis and clinical manifestations in the respective diarrheal diseases. In this review, the results of the morphological analyses of these patterns over the last 3 decades as well as some methodological problems encountered in the interpretation of morphological observations are discussed.     

Molecular methods for serovar determination of Salmonella

Abstract

Salmonella is a diverse foodborne pathogen, which has more than 2600 recognized serovars. Classification of Salmonella isolates into serovars is essential for surveillance and epidemiological investigations; however, determination of Salmonella serovars, by traditional serotyping, has some important limitations (e.g. labor intensive, time consuming). To overcome these limitations, multiple methods have been investigated to develop molecular serotyping schemes. Currently, molecular methods to predict Salmonella serovars include (i) molecular subtyping methods (e.g. PFGE, MLST), (ii) classification using serovar-specific genomic markers and (iii) direct methods, which identify genes encoding antigens or biosynthesis of antigens used for serotyping. Here, we reviewed reported methodologies for Salmonella molecular serotyping and determined the “serovar-prediction accuracy”, as the percentage of isolates for which the serovar was correctly classified by a given method. Serovar-prediction accuracy ranged from 0 to 100%, 51 to 100% and 33 to 100% for molecular subtyping, serovar-specific genomic markers and direct methods, respectively. Major limitations of available schemes are errors in predicting closely related serovars (e.g. Typhimurium and 4,5,12:i:-), and polyphyletic serovars (e.g. Newport, Saintpaul). The high diversity of Salmonella serovars represents a considerable challenge for molecular serotyping approaches. With the recent improvement in sequencing technologies, full genome sequencing could be developed into a promising molecular approach to serotype Salmonella.     

基于上转发光免疫层析技术的常见食源性致病菌快速检测方法研究与评价

目的：研制针对4种常见食源性致病菌：甲型副伤寒沙门菌（ Salmonella paratyphi A）、乙型副伤寒沙门菌（Salmonella paratyphi B）、大肠埃希菌O157∶H7（Escherichia coli O157∶H7）、副溶血弧菌（Vibrio parahaemolyticus）的基于上转发光免疫层析技术（ UPT－LF）的快速定量检测试纸,并对其检测性能进行评价。方法以上转发光纳米颗粒（ UCP－NPs）作为示踪物,基于双抗体夹心检测模型,研制针对上述4种靶标菌的UPT－LF试纸;以4种靶标菌的系列浓度标准菌悬液作为标准样品,评价试纸的敏感性、特异性、线性和精密性,并进行模拟染菌食品的检测,评价模拟样品阳性检出率。结果针对4种靶标菌的UPT－LF试纸均具有良好的线性,相关系数r为0．985～0．996;检测敏感性达105～106 CFU／ml,与其他近缘菌株无交叉反应;对乳制品、饮料、小食品、水产、肉类等细菌污染的食品检出率较高。结论该研究建立的对4种常见食源性致病菌进行快速检测的UPT－LF,简便快速,具有良好的敏感性、特异性和线性定量能力,操作性能可满足食品安全检测的要求。     

Membrane voltage-dependent activation mechanism of the bacterial flagellar protein export apparatus

The proton motive force (PMF) consists of the electric potential difference (Δψ), which is measured as membrane voltage, and the proton concentration difference (ΔpH) across the cytoplasmic membrane. The flagellar protein export machinery is composed of a PMF-driven transmembrane export gate complex and a cytoplasmic ATPase ring complex consisting of FliH, FliI, and FliJ. ATP hydrolysis by the FliI ATPase activates the export gate complex to become an active protein transporter utilizing Δψ to drive proton-coupled protein export. An interaction between FliJ and a transmembrane ion channel protein, FlhA, is a critical step for Δψ-driven protein export. To clarify how Δψ is utilized for flagellar protein export, we analyzed the export properties of the export gate complex in the absence of FliH and FliI. The protein transport activity of the export gate complex was very low at external pH 7.0 but increased significantly with an increase in Δψ by an upward shift of external pH from 7.0 to 8.5. This observation suggests that the export gate complex is equipped with a voltage-gated mechanism. An increase in the cytoplasmic level of FliJ and a gain-of-function mutation in FlhA significantly reduced the Δψ dependency of flagellar protein export by the export gate complex. However, deletion of FliJ decreased Δψ-dependent protein export significantly. We propose that Δψ is required for efficient interaction between FliJ and FlhA to open the FlhA ion channel to conduct protons to drive flagellar protein export in a Δψ-dependent manner.

The ion motive force (IMF) across the cell membrane is one of the most important sources of biological energy in any cell. The IMF is utilized for many essential biological activities, such as ATP synthesis, solute transport, nutrient uptake, protein secretion, flagella-driven motility, and so on (1). The IMF is the sum of the electrical (Δψ) and chemical (ΔpI) potential differences of ions such as protons (H⁺) (the proton motive force [PMF]) and sodium ions (Na⁺) (the sodium motive force [SMF]) across the membrane and is defined by Eq. 1:IMF=Vm+kBTq ln[ion]in[ion]ex,[1]where V_m is Δψ; [ion]_in and [ion]_ex are the internal and external ion concentrations, respectively; k_B is Boltzmann’s constant; T is the absolute temperature (in kelvins); and q is the charge of the ion. The Δψ corresponds to the membrane voltage (2).The flagellum of the enteric bacterium Salmonella enterica serovar Typhimurium (hereafter referred to as Salmonella) is a supramolecular motility machine consisting of the basal body, which acts as a bidirectional rotary motor; the hook, which functions as a universal joint; and the filament, which works as a helical propeller. The Salmonella flagellar motor is powered by a PMF across the cytoplasmic membrane. The motor consists of a rotor and multiple stator units, each of which acts as a transmembrane proton channel complex. The stator unit converts the proton influx through the channel into the force for high-speed rotation of the long helical filament (3, 4).For construction of the hook and filament structures at the cell exterior, a specialized protein transporter utilizes the PMF to transport flagellar building blocks to the distal end of the growing flagellar structure. The flagellar protein transporter consists of a PMF-driven export gate complex made of five transmembrane proteins, FlhA, FlhB, FliP, FliQ, and FliR, and an ATPase ring complex consisting of three cytoplasmic proteins, FliH, FliI, and FliJ (SI Appendix, Fig. S1) (5, 6). These proteins are evolutionarily related to those of the virulence-associated type III secretion systems of pathogenic bacteria, which inject effector proteins into eukaryotic host cells for invasion (7). Furthermore, the entire structure of the ATPase ring complex is structurally similar to the cytoplasmic F₁ part of F_OF₁-ATP synthase, which utilizes the PMF for ATP synthesis (8–10).FliI forms a homo-hexamer that hydrolyzes ATP at an interface between neighboring FliI subunits (10–12). FliJ binds to the central pore of the FliI ring (9). ATP hydrolysis by the FliI ATPase not only activates the transmembrane export gate complex through an interaction between FliJ and the C-terminal cytoplasmic domain of FlhA (FlhA_C) (13, 14) but also opens the entrance gate of the polypeptide channel through an interaction between FliI and the C-terminal cytoplasmic domain of FlhB (FlhB_C) (15). As a result, the export gate complex becomes an active proton/protein antiporter that couples an inward-directed H⁺ flow with an outward-directed protein export (SI Appendix, Fig. S1) (16). When the cytoplasmic ATPase complex becomes nonfunctional, the FlgN chaperone activates the Na⁺-driven export engine of the export gate complex over a wide range of external pH, allowing the export gate complex to drive Na⁺-coupled protein export (17, 18). The transmembrane domain of FlhA (FlhA_TM) acts as a transmembrane ion channel for the transit of both H⁺ and Na⁺ across the cytoplasmic membrane (17).A chemical potential gradient of either H⁺ (ΔpH) or Na⁺ (ΔpNa) is required for efficient inward-directed translocation of H⁺ or Na⁺ when FliH and FliI are absent (13, 17). Although the Δψ component is critical for flagellar protein export by the wild-type export gate complex (19), it remains unknown when and how Δψ is used for the flagellar protein export process. To clarify this question, we used the Salmonella MMHI0117 [ΔfliH-fliI flhB(P28T)] strain (hereafter referred to as ΔHI B*; Table 1) (20), in which the export gate complex uses both Δψ and ΔpNa at different steps of the flagellar protein export process (13, 17). We show that an increase in Δψ generated by an upward shift of the external pH from 7.0 to 8.5 activates flagellar protein export by this mutant even in the absence of ΔpNa, suggesting the presence of a Δψ-dependent activation mechanism for proton-coupled protein secretion by the export gate complex. We also show that an increased Δψ facilitates efficient docking of FliJ to FlhA_C.Table 1.Summary for flagellar protein export properties of Salmonella strains used in this study

Amino acids 89–96 of Salmonella typhimurium flagellin represent the major domain responsible for TLR5-independent adjuvanticity in the humoral immune response

Toll-like receptor 5 (TLR5) signaling in response to flagellin is dispensable for inducing humoral immunity, but alterations of aa 89–96, the TLR5 binding site, significantly reduced the adjuvanticity of flagellin. These observations indicate that the underlying mechanism remains incompletely understood. Here, we found that the native form of Salmonella typhimurium aa 89–96-mutant flagellin extracted from flagella retains some TLR5 recognition activity, indicating that aa 89–96 is the primary, but not the only site that imparts TLR5 activity. Additionally, this mutation impaired the production of IL-1β and IL-18. Using TLR5KO mice, we found that aa 89–96 is critical for the humoral adjuvant effect, but this effect was independent of TLR5 activation triggered by this region of flagellin. In summary, our findings suggest that aa 89–96 of flagellin is not only the crucial site responsible for TLR5 recognition, but is also important for humoral immune adjuvanticity through a TLR5-independent pathway.